A Novel Selection Method Based on the Expression Level of Green Fluorescent Protein Measured with a Quantitative Fluorescence Imager

Abstract

Green fluorescent protein (GFP) has been used extensively as a novel non-invasive reporter in the investigation of issues such as promoter function, transformation marker recognition, and sub-cellular localization analysis. A plant-optimized synthetic GFP modified via the replacement of the serine to threonine at position 65 [sGFP(S65T)] has achieved high levels of expression with no toxic side-effects in many plants. It would be useful to develop a non-invasive selection system based only upon GFP expression levels. A trapping vector was constructed using an sGFP(S65T) and a quantitative fluorescence imaging system was used for non-invasive screening of trapped tobacco cultured cells. Putative trapped lines could be identified using this system. Moreover, the GFP products from the candidate trapped lines could be quantitatively visualized directly via SDS-PAGE using the fluorescence imaging system. We conclude that the non-invasive selection system described here could be a powerful new tool for plant biotechnology.